Highly-purified recombinant reverse transcriptase

Abstract

A plasmid for expression of Moloney Murine Leukemia Virus-derived reverse transcriptase in E. coli cells deficient in the expression of indiginous RNAse activity, a method for purification of the recombinant enzyme, and a composition comprising a cloned and purified reverse transcriptase opimized for use in cDNA and nucleic acid amplification procedures.

What is claimed is: 
     
       1. A method for purifying a recombinant polypeptide having RNA-directed and DNA-directed DNA polymerase activities, comprising the steps of: providing a plurality of Escherichia coli host cells having about 0.1% or less of wild type RNase I activity and capable of expressing a recombinant vector comprising a nucleic acid sequence derived from a Moloney murine leukemia virus sequence encoding a recombinant polypeptide having RNA-directed and DNA-directed DNA polymerase activities;   lysing a plurality of said host cells in which the recombinant polypeptide has been expressed, and removing cellular debris therefrom, thereby forming a cell lysate;   applying the cell lysate to a cation-exchange medium in the presence of a solution having a conductivity of no more than about 0.05 M NaCl, thereby binding said polypeptide to the cation-exchange medium;   then eluting the polypeptide from the cation-exchange medium by contacting said polypeptide with a salt gradient beginning with a conductivity of about 0.2 M NaCl and ending with a conductivity of about 0.7 M NaCl;   retaining at least one fraction containing the polypeptide;   applying the fraction containing the polypeptide to a gel filtration column; and   recovering at least one fraction containing said polypeptide.   
     
     
       2. The method of claim 1 wherein the polypeptide has an apparent molecular weight of about 70,000 daltons. 
     
     
       3. The method of claim 1 wherein the host cells are selected from the group consisting of Escherichia coli strains 1200, MRE 600, Q13, and A 19. 
     
     
       4. The method of claim 3 wherein said polypeptide is the same length as mature Moloney murine leukemia virus reverse transcriptase. 
     
     
       5. The method of claim 3, wherein the host cells are Escherichia coli strain 1200.